The long-term objective of this proposal is to determine the effects of chronic ethanol on a system in the liver that plays a major role in hepatic energy metabolism, namely the mitochondrion. Studies related to this goal will be implemented to establish if ethanol-induced oxidative stress alters hepatic energy metabolism. Analyses will be performed to determine if ethanol-induced reactive oxygen species oxidatively modify mitochondrial proteins. Specifically, these investigations will be carried out to determine if ethanol-induced reactive oxygen species: (1) increase oxidative modification of proteins within the isolate mitochondrion; (2) oxidatively modify and subsequently inactivate the selenium-dependent mitochondrial glutathione peroxidase enzyme; (3) depress hepatic mitochondrial protein synthesis by oxidative modification of the hepatic mitochondrial ribosomal proteins; and (4) decrease the function of the oxidative phosphorylation system by oxidative modification of the mitochondrial electron transport chain proteins and the ATP synthase. To address these Specific Aims, the effects of acute and chronic ethanol will be studied. Acute ethanol effects will be examined by using intragastric ethanol administration (4.0 g ethanol/kg body weight). For chronic studies, rats will be fed liquid diets in which ethanol comprises 36% of total calories for 31 days. The chronic ethanol-related oxidative modification of proteins in mitochondria will be measured using the 2,4-diniphenylhydrazine assay for the determination of protein carbonyl groups. Levels of oxidized mitochondrial protein will be correlated with the concentration of reactive oxygen species generated in the mitochondrion. Levels of reactive oxygen species will be measured using the dichlorogluorescein assay. Ethanol-induced alterations in the activity and protein level of mitochondrial glutathione peroxidase will be measured after acute and chronic ethanol exposure. Analysis of glutathione peroxidase protein oxidation will be performed using a novel Western blot immunoassay that detects 2,4-dinitrophenylhydrazine- reactive protein carbonyl groups. Oxidized mitochondrial ribosomal proteins and electron transport chain proteins will be analyzed using this Western blot immunoassay after acute and chronic ethanol exposure. Oxidized mitochondrial electron transport chain proteins will be identified.